CN114058533A - Screening method of sulfate reducing bacteria capable of degrading polycyclic aromatic hydrocarbons - Google Patents
Screening method of sulfate reducing bacteria capable of degrading polycyclic aromatic hydrocarbons Download PDFInfo
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Abstract
The invention provides a method for screening sulfate reducing bacteria capable of degrading polycyclic aromatic hydrocarbons, which comprises the following steps: carrying out enrichment culture on a sample containing sulfate reducing bacteria to obtain enriched bacterial liquid, and then carrying out anaerobic culture on the enriched bacterial liquid by a dish stacking sandwich method; in the dish-stacked sandwich method, a solid culture medium contains sodium sulfite and L-cysteine hydrochloride, and polycyclic aromatic hydrocarbon and the enriched bacterial liquid are coated under anaerobic conditions. The method can efficiently obtain the anaerobic degradation strain with PAHs degradation capability, and the method is simple to operate and has wide application prospect.
Description
Technical Field
The invention relates to the technical field of environmental microorganisms, in particular to a method for screening sulfate reducing bacteria capable of degrading polycyclic aromatic hydrocarbons.
Background
Polycyclic Aromatic Hydrocarbons (PAHs) are mainly substances with strong toxicity generated by petroleum pollution, oil tanker leakage, automobile exhaust, incomplete combustion of natural fuels such as coal and petroleum, and are toxic organic pollutants formed by connecting two or more benzene rings through thick bonds. The PAHs have stable chemical structures and strong hydrophobicity, so that the PAHs are widely distributed and stably exist in the environment, and the PAHs with high molecular weight are easily accumulated in a large amount. Meanwhile, the fat solubility of the PAHs makes the PAHs easily absorbed by animals and difficult to discharge, and the PAHs can be enriched in animals and plants step by step through a food chain. Most PAHs have the effect of causing three causes (carcinogenesis, teratogenesis and mutagenesis) and have great potential harm to human health and ecological environment. Therefore, in recent years, researchers have attracted extensive attention to removing the pollution of PAHs in the environment.
PAHs have strong chemical stability and high removal difficulty, and the microbial degradation technology has the advantages of low treatment cost and quick response both ecologically and economically, and is concerned at home and abroad. Phenanthrene is a model compound for researching biodegradation of PAHs, and aerobic degradation strains with phenanthrene degradation capability have been reported in recent decades, including Rhodococcus (Rhodococcus), Pseudomonas (Pseudomonas), Vibrio (Vibrio), Bacillus (Bacillus) and Micrococcus (Micrococcus). However, the underground aquifer or contaminated soil is typically anaerobic, allowing aerobic microorganisms in the contaminated zone to be replaced by anaerobic microorganisms. Thus, anaerobic biodegradation of PAHs is more feasible and cost effective than aerobic biodegradation.
Sulfate-Reducing bacteria (SRB, the same below) are microorganisms that can dissimilate Sulfate, other oxidized sulfides, and elemental sulfur as electron acceptors, and are widely present in our environment, including deep soil, paddy soil, marine sediments, groundwater, and sulfur-containing sediments, and thus SRB can be used to remediate pollutants in various anaerobic environments, including PAHs. SRB plays an important role in the remediation of soil and groundwater in contaminated anaerobic areas.
SRB is screened widely by a plurality of researchers due to important functions thereof, however, most SRB obtained by the existing screening is Desulfovibrio (Desulfovibrio), the sensitivity to oxygen and oxidation-reduction potential is not high, the PAHs in an anaerobic area cannot be effectively degraded, and the further development and application research of anaerobic bioremediation technology of PAHs polluted soil and underground water in the anaerobic area are restricted. Therefore, the separated and domesticated PAHs anaerobic degrading bacteria have important significance for treating and restoring PAHs polluted soil and underground water.
Disclosure of Invention
Therefore, after a great deal of research on a method for screening sulfate reducing bacteria capable of anaerobically degrading polycyclic aromatic hydrocarbons, the inventor finds that PAHs anaerobic degrading bacteria cannot be screened by the existing method, and the key reason is that a culture medium (such as a culture medium added with L-cysteine, ascorbic acid, ferrous sulfate and other components) in the existing method only can reduce the oxidation-reduction potential of the culture medium, and the culture medium still contains trace oxygen and does not contain PAHs, so that strict anaerobic degrading bacteria of PAHs cannot be screened.
Specifically, the invention provides a method for screening sulfate reducing bacteria capable of degrading polycyclic aromatic hydrocarbons, which comprises the following steps: carrying out enrichment culture on a sample containing sulfate reducing bacteria to obtain enriched bacterial liquid, and then carrying out anaerobic culture on the enriched bacterial liquid by a dish stacking sandwich method;
in the dish-stacked sandwich method, a solid culture medium contains sodium sulfite and L-cysteine hydrochloride, and polycyclic aromatic hydrocarbon and the enriched bacterial liquid are coated under anaerobic conditions.
The invention discovers that after the solid culture medium used in the laminated dish sandwich method and the conditions for coating the bacteria liquid are optimized, the oxidation-reduction potential of the culture medium is reduced, trace oxygen in the culture medium is removed, the growth environment of the SRB is greatly improved, and the anaerobic SRB with PAHs degradation capability can be efficiently separated.
Preferably, the sodium sulfite is added in the following mode: under the anaerobic condition, sodium sulfite solution with the concentration of 50-150g/L is dripped into a solid culture medium to ensure that the dissolved oxygen is zero and the oxidation-reduction potential is less than-100 mV; the concentration of the sodium sulfite solution is preferably 100. + -.10 g/L.
In a specific embodiment, the sodium sulfite solution is dropwise added into the solid culture medium containing resazurin, and when the color of the solution becomes colorless, the oxygen solubility and the redox potential in the solid culture medium are shown to meet the requirements.
Preferably, the addition mode of the L-cysteine hydrochloride is as follows: under anaerobic conditions, adding an L-cysteine hydrochloride solution with the concentration of 50-150g/L into the solid culture medium, wherein the addition amount of the L-cysteine hydrochloride solution is 2 +/-0.5 mL by taking 1L of the solid culture medium as a reference. The concentration of the L-cysteine hydrochloride solution is preferably 100 + -10 g/L. Thereby, the oxidation-reduction potential of the solid medium was further lowered to-200 mV.
The invention further discovers that the sodium sulfite and the L-cysteine hydrochloride are added into the solid culture medium according to the mode, so that residual trace oxygen in the solid culture medium can be efficiently removed, the oxidation-reduction potential of the solid culture medium is reduced to an extremely low level, the screening and separation period is shortened, and meanwhile, the screening of anaerobic SRB with PAHs degradation capability is facilitated.
Preferably, the polycyclic aromatic hydrocarbon comprises one or more of naphthalene, phenanthrene, benzo [ a ] anthracene, acenaphthene, fluorene and pyrene; phenanthrene is preferred.
Preferably, the sample is petroleum-contaminated bottom soil; preferably soil below 20cm above ground. The content of sulfate-reducing bacteria in the sample can be determined by the person skilled in the art based on the general knowledge.
Preferably, the enrichment culture comprises: and under the anaerobic condition, adding the sample into a liquid culture medium, and carrying out enrichment culture to obtain an enriched bacterial liquid.
Preferably, the preparation method of the liquid culture medium comprises the following steps: reacting NH4Cl 0.5-0.7g/L，KH2PO4 0.5-0.7g/L，K2HPO4 0.6-0.8g/L，Na2SO42-3.5g/L, sterilizing with high pressure steam at 121 deg.C for 20 min; then adding CaCl280-120g/L，MgCl2 180-220g/L，FeCl240-60g/L and Na2S5-15 g/L solution 1 + -0.2 mL, NaHCO34 plus or minus 0.5mL of 40-60g/L solution, 10 plus or minus 1mL of trace element solution and 2 plus or minus 0.5mL of vitamin solution;
wherein the trace element solution comprises the following components:
CoCl2·6H2O 20-40mg/L，CuCl2 0.1-0.2mg/L，H3BO3 5-6mg/L，MnCl2·4H2O 18-22mg/L，Na2MO4·H2O 2-3mg/L，NiCl2·2H2O 1-2mg/L，ZnCl2 1.5-2.5mg/L；
the vitamin solution comprises the following components:
35-45mg/L of Biotin, 35-45mg/L of Folic acid, 220mg/L of Pyridorine Hd 180, 90-110mg/L of Riboflavin, 90-110mg/L of Tlimine, 90-110mg/L of Nicotinic acid, 90-110mg/L of Panto the amino acid, 90-15 mg/L of vitamin B10, 90-110mg/L of p-aminobenzoic acid and 90-110mg/L of thiooctanol;
the solid culture medium contains the liquid culture medium and 1.5-2 wt% of agar.
In the above-mentioned preparation process of the liquid medium and the solid medium, the skilled person can judge whether the sterilization of the respective components is required, and/or the sterilization method, according to the general knowledge.
Preferably, the screening method further comprises:
the colony obtained by the culture of the dish-stacked sandwich method is inoculated into a liquid culture medium containing polycyclic aromatic hydrocarbon for culture, and then the concentration of the residual polycyclic aromatic hydrocarbon in the liquid culture medium is measured and compared. Therefore, sulfate reducing bacteria with excellent PAHs degrading capability are screened out.
In some preferred embodiments, the concentration of residual polycyclic aromatic hydrocarbon in the liquid medium after 4-6 weeks of culture is determined and compared.
The person skilled in the art is able to know the method of determination and comparison of the concentration of residual polycyclic aromatic hydrocarbons in the liquid medium in accordance with common knowledge. Further preferably, the measurement method comprises: adding equal volume of dichloromethane into the liquid culture medium, extracting for 2-4min, and measuring by high performance liquid chromatography. The high performance liquid chromatography determination conditions are as follows: sample amount of 10. mu.L, C18 column, (85-95): methanol of 5-15): water is used as a mobile phase, the flow rate of the mobile phase is 0.5-1.5mL/min, the detection wavelength is 254nm, and the temperature of the column incubator is 40 +/-5.0 ℃.
The comparison method comprises the following steps: the concentrations of residual polycyclic aromatic hydrocarbon in the liquid medium to which colonies were added and the liquid medium to which no colonies were added were compared at the same time. More specifically, the three residual amounts measured by the blank control group and the three residual amounts measured by the culture are finished by using a double-sample T test of minitab 17, and if the p value is less than 0.05, the concentrations of the polycyclic aromatic hydrocarbon residual between the blank control group and the culture are considered to be obviously different, and the corresponding culture has the polycyclic aromatic hydrocarbon degradation capability.
Preferably, the screening method further comprises: and purifying and identifying the types of the colonies obtained by screening.
The method for identifying the purity and the type comprises the following steps: extracting DNA of a culture by using a Tiangen bacterium genome DNA extraction kit, amplifying 16S rRNA, sending to Beijing Saimer Lily Biotechnology Co., Ltd for sequencing, judging whether the culture is pure bacteria or not by judging whether a sequence obtained by sequencing is a unimodal sequence or not and a scanning electron microscope picture of the culture, and obtaining the type of the culture according to the comparison result of the sequencing sequence and a sequence in an NCBI database.
The 16S rRNA amplification method comprises the following steps: the 16S rRNA region of the bacteria was amplified using a PCR instrument using primers 27F and 1492R, and the PCR procedure was: pre-denaturation at 95 + -2 deg.C for 4-6min, then performing 30 amplification cycles, each cycle comprising denaturation at 95 + -2 deg.C for 40-80sec, annealing at optimal temperature for 40-80sec, extension at 72 + -2 deg.C for 40-80sec, final extension at 72 + -2 deg.C for 4-6min after all cycles are completed, and final storage at 4 deg.C until use. Each sample was PCR-replicated three times, and the three PCR products were mixed together and then verified for the length of the amplified sequence by agarose electrophoresis.
Preferably, the anaerobic conditions are achieved by anaerobic bottles and/or anaerobic glove boxes.
Specifically, the invention provides a method for screening sulfate reducing bacteria capable of degrading polycyclic aromatic hydrocarbons, which comprises the following steps:
1) under the anaerobic condition, adding bottom soil polluted by petroleum into a liquid culture medium, and carrying out enrichment culture to obtain an enriched bacterial liquid;
2) then carrying out anaerobic culture on the enriched bacterial liquid by a dish stacking sandwich method, wherein in the dish stacking sandwich method, a used solid culture medium contains sodium sulfite and L-cysteine hydrochloride, and polycyclic aromatic hydrocarbon and the enriched bacterial liquid are coated in an anaerobic condition, preferably in an anaerobic glove box;
3) inoculating the bacterial colony obtained by the culture of the laminated dish sandwich method into a liquid culture medium containing polycyclic aromatic hydrocarbon for culture, and then measuring and comparing the concentration of the residual polycyclic aromatic hydrocarbon in the liquid culture medium;
4) and purifying and identifying the types of the colonies obtained by screening.
In order to reduce the contact time of the sample and oxygen and shorten the enrichment culture time, the invention preferably carries out the following pretreatment on the sample:
the samples were loaded into an anaerobic incubator, then placed into a foam box containing dry ice, transported to a laboratory, and immediately transferred into an anaerobic glove box, and the samples were ground by sieving with a 200 mesh sieve.
Preferably, in the enrichment culture: the weight volume ratio of the sample to the liquid culture medium is (0.5-1) g: 50 ml;
the conditions of the enrichment culture comprise: culturing at 28-32 deg.C under dark condition and 200rpm at 100-35 days, transferring to new liquid culture medium at 5-10% inoculum size for 3-5 times.
The invention also provides sulfate reducing bacteria capable of degrading polycyclic aromatic hydrocarbons, which are obtained by the screening method.
Based on the technical scheme, the invention has the following beneficial effects:
the method further reduces a small amount of residual dissolved oxygen in the solid culture medium, avoids the residual dissolved oxygen from temporarily contacting with oxygen in the air when the sulfate reducing bacteria are coated, and simultaneously solves the problem that the oxidation-reduction potential of the environment contacting with one side of the sulfate reducing bacteria in the air is not low enough, so that the anaerobic degradation strain with PAHs degradation capability can be efficiently obtained, and the method is simple to operate and has wide application prospect.
Drawings
Fig. 1 is a flow chart of the screening method of sulfate-reducing bacteria capable of degrading polycyclic aromatic hydrocarbons provided by the present invention.
FIG. 2 is a plate diagram of colonies screened by the method of the present invention.
FIG. 3 is a phylogenetic tree of sulfate-reducing pure bacteria PheS1 obtained by screening with the method provided by the present invention.
FIG. 4 is a phylogenetic tree of the sulfate-reducing pure bacteria PheS2 obtained by screening with the method provided by the invention.
FIG. 5 is a phylogenetic tree of sulfate-reducing pure bacteria S8 screened by the method provided in comparative example 1.
FIG. 6 shows phenanthrene degradation of sulfate-reducing bacteria screened by the method of the present invention and sulfate-reducing bacteria screened by the method of comparative example 1.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.
The examples do not show the specific techniques or conditions, according to the technical or conditions described in the literature in the field, or according to the product specifications. The reagents or instruments used are conventional products available from regular distributors, not indicated by the manufacturer.
Example 1
As shown in FIG. 1, the screening method of sulfate reducing bacteria capable of anaerobically degrading phenanthrene comprises the following steps: enriching phenanthrene anaerobic biodegradable sulfate reducing flora, combining strain dilution coating-anaerobic glove box and dish stacking sandwich method for screening, separating and purifying strains, measuring phenanthrene degrading and sulfate radical reducing capability of the screened sulfate reducing pure bacteria and preserving strains.
Enrichment of phenanthrene anaerobic biodegradable sulfate-reducing flora:
adding 50mL of basic culture medium into a 300mL small-mouth anaerobic bottle, vacuumizing for 1min, aerating high-purity nitrogen for 15min, sealing the anaerobic bottle with a butyl rubber plug, sterilizing at 121 deg.C under high pressure for 20min, transferring the anaerobic bottle into an anaerobic glove box, removing butyl rubber plug, adding 25mg/L phenanthrene, placing in an open air until the color of liquid culture medium changes from pink to colorless, adding oxygen-free inorganic salt ion filtered by sterilized 0.22 μm membrane, trace elements and vitamins to obtain liquid culture medium, then 0.5g of soil sample which passes through a 200-mesh screen and is in a petroleum-polluted area of the victory oil field is inoculated in the anaerobic bottle, the anaerobic bottle is sealed by a butyl rubber plug again, the anaerobic bottle is transferred out of the anaerobic glove box, culturing for one month in a light-proof shaking table at 30 ℃ and 150rpm, and judging that the enrichment is successful when the enrichment solution shows the ink color. The culture medium with 5% (v/v) total volume was transferred to fresh culture medium and repeated 5 times.
The preparation method of the liquid culture medium comprises the following steps: reacting NH4Cl 0.6g/L，KH2PO4 0.64g/L，K2HPO4 0.7g/L，Na2SO42.84g/L, 1L of distilled water, and high-pressure steam sterilization at 121 ℃ for 20 min. Then adding CaCl filtered through a sterilized 0.22 mu m filter membrane2 100g/L，MgCl2 200g/L，FeCl250g/L and Na2S10 g/L solution 1mL, NaHCO34mL of 50g/L solution, 10mL of trace element solution and 2mL of vitamin solution; wherein:
the formula of the trace element solution is as follows: CoCl2·6H2O 30mg/L，CuCl2 0.15mg/L，H3BO3 5.7mg/L，MnCl2·4H2O 20mg/L，Na2MO4·H2O 2.5mg/L，NiCl2·2H2O 1.5mg/L，ZnCl2 2.1mg/L；
The formula of the vitamin solution is as follows: biotin 40mg/L, Folic acid 40mg/L, Pyridorine Hd 200mg/L, Riboflavin 100mg/L, Tlimine 100mg/L, Nicotinic acid 100mg/L, Panto the acid 100mg/L, vitamin B122 mg/L, p-aminobenzoic acid 100mg/L, and thiocinol 100 mg/L.
The strain is subjected to dilution coating-anaerobic glove box and dish stacking sandwich method combined screening:
and adding 1.7 wt% of agar powder into the liquid culture medium to prepare a solid culture medium. Sterilizing the solid culture medium at 121 deg.C under high pressure for 20min, transferring into anaerobic glove box, removing residual dissolved oxygen in the solid culture medium by adding 200 μ L of 100g/L sodium sulfite solution (adding sodium sulfite until the color of the culture medium containing resazurin is changed from pink to colorless), adding 2mL of 100 g/L-cysteine hydrochloride solution into each liter of solid culture medium to further reduce the oxidation-reduction potential of the solid culture medium, pouring the solid culture medium into multiple plates, uniformly coating 0.5mg phenanthrene on the surface after the solid culture medium is solidified, coating 100 μ L of enriched flora in each dilution multiple in the anaerobic glove box, transferring another solid culture medium stored in a 90 deg.C oven into anaerobic glove box, pouring onto the solid culture medium coated with microorganism after the temperature of the solid culture medium is reduced to 55 deg.C, after the upper solid medium solidified, the upper cover of the plate was closed, and the plate was transferred into an incubator in an anaerobic glove box and incubated at 30 ℃ in the absence of light for 4 weeks. And removing the upper solid culture medium, and selecting black bacterial colonies with clear edges to inoculate the black bacterial colonies to a plurality of bottles of liquid culture medium containing phenanthrene.
Separating and purifying the strains:
extracting DNA of a culture by using a Tiangen bacterium genome DNA extraction kit, amplifying 16S rRNA, sending to Beijing Saimer Lily Biotechnology Co., Ltd for sequencing, judging whether a cultured microorganism is a pure bacterium or not by judging whether a sequence obtained by sequencing is a unimodal sequence or not, and obtaining the type of the cultured microorganism according to the comparison result of the sequencing sequence and the sequence in an NCBI database. The 16S rRNA amplification method comprises the following steps: the 16S rRNA region of the bacteria was amplified using a PCR instrument using primers 27F and 1492R, and the PCR procedure was: pre-denaturation at 95 ℃ for 5min, then entering 30 amplification cycles, each cycle comprising denaturation at 95 ℃ for 60sec, annealing at optimal temperature for 60sec, extension at 72 ℃ for 60sec, and finally extension at 72 ℃ for 5min after completion of all cycles, and finally storage at 4 ℃ until use. Each sample was PCR-replicated three times, and the three PCR products were mixed together and then verified for the length of the amplified sequence by agarose electrophoresis. If the culture is not pure, the new dilution coating-anaerobic glove box and dish sandwich method combined screening method is used for carrying out secondary, third or even more screening on the culture until the culture is pure bacteria.
And (3) measuring the phenanthrene degrading and sulfate radical reducing capacities of the screened pure sulfate reducing bacteria:
the residual phenanthrene in the solution was extracted by equal volume of dichloromethane for 3min in a whole bottle and then determined by high performance liquid chromatography, as obtained by comparing the concentration of the residual phenanthrene in the pure bacterial culture after 6 weeks of culture and the blank control without organisms. The high performance liquid chromatography determination conditions are as follows: sample size of 10. mu.L, C18 column, 90:10 methanol: water is used as a mobile phase, the flow rate of the mobile phase is 1mL/min, the detection wavelength is 254nm, and the temperature of a column oven is 40 ℃. The comparison method of the residual phenanthrene concentration is that the three residual amounts measured by the blank control group and the three residual amounts measured by the culture are finished by using a double-sample T test of minitab 17, if the p value is less than 0.05, the residual phenanthrene concentrations of the blank control group and the pure bacteria culture are considered to have obvious difference, and the corresponding pure bacteria have the capacity of degrading phenanthrene. The sulfate radical concentration is determined by anion chromatography, and the sulfate radical reducing ability of the strain is judged according to the change of the sulfate radical concentration.
And (3) preservation of strains:
the inclined tube with the cultured mycelial bacterial colony is obtained by an inclined tube puncturing culture method and then is stored in a refrigerator at the temperature of 80 ℃ below zero.
Comparative example 1
This comparative example provides a screening method of sulfate-reducing bacteria, which is different from example 1 only in that only L-cysteine hydrochloride was added to the solid medium, that is, 2.5mL of a 100 g/L-cysteine hydrochloride solution was added per liter of the solid medium to lower the oxidation-reduction potential of the solid medium to less than-200 mV.
Test examples
The strains screened in example 1 and comparative example 1 were subjected to degradation tests of polycyclic aromatic hydrocarbons, respectively.
The specific method comprises the following steps: respectively inoculating the strains screened in the example 1 and the comparative example 1 into a liquid culture medium containing phenanthrene with the concentration of 20-30mg/L, extracting an initial sample and a culture by using dichloromethane with the same volume after culturing for 6 weeks, then determining the concentration of the phenanthrene by using high performance liquid chromatography, and judging whether the screened strains have the phenanthrene degradation capacity or not by using the change of the concentration of the phenanthrene.
The sulfate-reducing strain PheS1 screened in example 1 was used to degrade phenanthrene at 78% of the initial concentration of 22.7mg/L in 6 weeks, and the sulfate-reducing strain PheS2 was used to degrade phenanthrene at 99% of the initial concentration of 22.9mg/L in 6 weeks. And the degradation rate of the sulfate-reducing strain S8 screened by the comparative example 1 to phenanthrene with the initial concentration of 22.6mg/L in 6 weeks is almost zero.
Meanwhile, the invention verifies the polycyclic aromatic hydrocarbon degradation effect of sulfate reducing bacteria screened and separated in the patent document CN 107058108B, namely the content of CN 107058108B example 1 is repeated (wherein the related process parameters are all right-side end values). The results show that the strains obtained by screening and separation according to the method described in CN 107058108B do not have the ability of degrading phenanthrene.
The results show that: the strains screened by the method have the capacity of degrading the polycyclic aromatic hydrocarbon.
Finally, it should be noted that: the above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions of the embodiments of the present invention.
Claims (10)
1. A method for screening sulfate reducing bacteria capable of degrading polycyclic aromatic hydrocarbons comprises the following steps: carrying out enrichment culture on a sample containing sulfate reducing bacteria to obtain enriched bacterial liquid, and then carrying out anaerobic culture on the enriched bacterial liquid by a dish stacking sandwich method; it is characterized in that the preparation method is characterized in that,
in the dish-stacked sandwich method, a solid culture medium contains sodium sulfite and L-cysteine hydrochloride, and polycyclic aromatic hydrocarbon and the enriched bacterial liquid are coated under anaerobic conditions.
2. The screening method according to claim 1, wherein the sodium sulfite is added in a manner of: under the anaerobic condition, sodium sulfite solution with the concentration of 50-150g/L is dripped into a solid culture medium to ensure that the dissolved oxygen is zero and the oxidation-reduction potential is less than-100 mV;
the concentration of the sodium sulfite solution is preferably 100. + -.10 g/L.
3. Screening method according to claim 1 or 2, characterized in that the L-cysteine hydrochloride is added in the form of: under anaerobic conditions, adding an L-cysteine hydrochloride solution with the concentration of 50-150g/L into the solid culture medium, wherein the addition amount of the L-cysteine hydrochloride solution is 2 +/-0.5 mL by taking 1L of the solid culture medium as a reference.
The concentration of the L-cysteine hydrochloride solution is preferably 100 + -10 g/L.
4. The screening method according to any one of claims 1 to 3, wherein the polycyclic aromatic hydrocarbon includes one or more of naphthalene, phenanthrene, benzo [ a ] anthracene, acenaphthene, fluorene, pyrene; preferably phenanthrene;
and/or the sample is petroleum-contaminated bottom soil; preferably soil below 20cm above ground.
5. The screening method according to any one of claims 1 to 4, wherein the enrichment culture comprises: and under the anaerobic condition, adding the sample into a liquid culture medium, and carrying out enrichment culture to obtain an enriched bacterial liquid.
6. The screening method according to claim 5,
the preparation method of the liquid culture medium comprises the following steps: will contain NH4Cl 0.5-0.7g/L，KH2PO4 0.5-0.7g/L，K2HPO4 0.6-0.8g/L，Na2SO4Carrying out high-pressure steam sterilization on 2-3.5g/L of a basic culture medium; then adding CaCl into 1L basal medium2 80-120g/L，MgCl2 180-220g/L，FeCl240-60g/L and Na2S5-15 g/L solution 1 + -0.2 mL, NaHCO34 plus or minus 0.5mL of 40-60g/L solution, 10 plus or minus 1mL of trace element solution and 2 plus or minus 0.5mL of vitamin solution;
wherein the trace element solution comprises the following components:
CoCl2·6H2O 20-40mg/L，CuCl2 0.1-0.2mg/L，H3BO3 5-6mg/L，MnCl2·4H2O 18-22mg/L，Na2MO4·H2O 2-3mg/L，NiCl2·2H2O 1-2mg/L，ZnCl2 1.5-2.5mg/L；
the vitamin solution comprises the following components:
35-45mg/L of Biotin, 35-45mg/L of Folic acid, 220mg/L of Pyridorine Hd 180, 90-110mg/L of Riboflavin, 90-110mg/L of Tlimine, 90-110mg/L of Nicotinic acid, 90-110mg/L of Panto the amino acid, 10-15mg/L of vitamin B, 90-110mg/L of p-aminobenzoic acid and 90-110mg/L of thiooctanol;
the solid culture medium contains the liquid culture medium and 1.5-2 wt% of agar.
7. The screening method according to any one of claims 1 to 6, further comprising:
the colony obtained by the culture of the dish-stacked sandwich method is inoculated into a liquid culture medium containing polycyclic aromatic hydrocarbon for culture, and then the concentration of the residual polycyclic aromatic hydrocarbon in the liquid culture medium is measured and compared.
8. The screening method of claim 7, further comprising: and purifying and identifying the types of the colonies obtained by screening.
9. Screening method according to any one of claims 1 to 8, wherein said anaerobic conditions are achieved by means of anaerobic bottles and/or anaerobic glove boxes.
10. Sulfate-reducing bacteria capable of degrading polycyclic aromatic hydrocarbons, which are obtained by the screening method according to any one of claims 1 to 9.
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